Macrophages (M?) are involved in the pathogenesis of Acute Lung Injury (ALI), yet their potentially important role in resolving inflammatory lung injury is not well understood. In Project 2, we propose to investigate the role of a unique population of macrophages (M?) termed S1PM? identified by us that are capable of locally producing sphingosine-1-phosphate (S1P) in the inflammatory environment to resolve lung injury. Specifically, we will focus on the novel concept that generation of S1PM? occurs at the onset of the resolution phase of lung injury through PAR2-CREB1-mediated induction of SPHK2 expression. We postulate that this signaling cascade resulting in the generation of the S1PM? population in injured lungs is a fundamental mechanism for resolving lung injury. Thus, using a range of powerful approaches such as several novel genetic mouse models including macrophage depleted mice and mice where S1PM? generation can be traced , 2-photon imaging, deep analyses of relevant end-points, and samples from acute respiratory distress syndrome (ARDS) patients, we will extensively characterize the S1PM? population, the mechanisms regulating its generation in lungs, and the role of these cells in resolving inflammatory lung injury. To that end, we will carry out the following Aims: AIM 1: To define the population of SPHK2-induced S1PM? and their role in restoring lung vascular and alveolar epithelial barrier integrity, promoting resolution of inflammatory lung injury, and their potential for inducing tolerance to lung injury. In this aim, we will test the hypothesis that SPHK2 expression in M? induces the generation of the S1PM? population, which upon releasing S1P in injured lungs activates the S1P receptor1 (S1PR1) in endothelial cells and S1PR2 in type II alveolar epithelial cells to signal proliferation of these cells and re-annealing of alveolar-capillary barriers. Aim 2: To determine the role of PAR2 and activation of the downstream pathway involving the transcription factor CREB1 in signaling the generation of the S1PM? population and whether activation of this signaling is essential for resolving inflammatory lung injury. In this aim we will define the signaling pathways responsible for giving rise to the S1PM? population and determine the causal role of these cells in resolving inflammatory lung injury. We will also determine the function of PAR2 in transcriptionally regulating SPHK2 expression through CREB1 and address the concept that pharmacological activation of PAR2 following injury promotes resolution of inflammatory lung injury and define the underlying mechanisms of resolution and tolerance. Together with these comprehensive studies, and through interactions with other Projects of the Program, we will characterize and define the S1PM? population and their role in resolving inflammatory lung injury and promoting resolution to injury. Since M? are involved in the pathogenesis of ALI, we expect our studies to lay new conceptual groundwork for the development of novel therapeutic approaches based on the use of these cells for treatment of ALI.